Bacillus thuringiensis strains toxic to diabrotica species

ABSTRACT

Two new Bacillus thuringiensis strains, BTS02584B and BTS02584C, produce insecticidal components that are toxic to Coleoptera, more particularly toxic to Diabrotica spp. The strains themselves, their sporulated cultures, or their insecticidally effective components can be used as the active ingredient in an insecticidal composition for combatting Coleoptera, more specifically Diabrotica species.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to two new strains of Bacillusthuringiensis (the "BTS02584B and BTS02584C strains") which producecrystal proteins (the "BTS02584B and BTS02584C crystal proteins") whichare packaged in crystals (the "BTS02584B and BTS02584C crystals") duringsporulation.

The present invention also relates to an insecticide composition that isactive against Coleoptera, more particularly against Diabroticaspecies(hereinafter abbreviated "spp.") which comprises sporulatedcultures of the BTS02584B or BTS02584C strains or the activecomponent(s) thereof as an active insecticidal ingredient.

The present invention also relates to a method for combatting Diabroticapests by contacting these pests with BTS02584B and/or BTS02584C strains,sporulated cultures of the BTS02584B and/or BTS02584C strains or withtheir insecticidally effective component(s).

2. Description of the Prior Art

Some of the most destructive pests are found among the Diabroticinabeetles. In North America, the three important species of cornrootworms, Diabrotica virgifera virgifera (the Western corn rootworm),Diabrotica barberi (the Northern corn rootworm) and Diabroticaundecimpunctata howardi (the Southern corn rootworm) are considered tobe the most expensive insect pests to control (Metcalf, 1986).Diabrotica virgifera virgifera and Diabrotica barberi are considered themost serious insect pests of corn in the major corn-producing states ofthe United States and Canada (Levine and Oloumi-Sadeghi 1991). Thelarvae feed on the roots and thus cause direct damage to corn growth andcorn yields. Costs for soil insecticides to control larval damage to theroot systems of corn and aerial sprays to reduce beetle damage to cornsilks, when combined with crop losses, can approach one billion dollarsannually (Metcalf, 1986).

B. thuringiensis is a gram-positive bacterium which produces endogenouscrystals upon sporulation. The crystals are composed of proteins whichare specifically toxic against insect larvae. Three different Btpathotypes have been described. Pathotype A that is active againstLepidoptera; pathotype B that is active against certain Diptera, e.g.,mosquitoes and black flies; and pathotype C that is active againstColeoptera, e.g., beetles (Ellar et al., 1986). The fact thatconventional submerged fermentation techniques can be used to produce Btspores and sporulated cultures on a large scale makes Bt bacteriacommercially attractive as a source of insecticidal compositions.

A Bt strain, whose crystals are toxic to Coleoptera, has been describedas Bt tenebrionis and BTS1 in U.S. Pat. No. 4,766,203; European PatentPublication No. 0,213,818; U.S. Pat. No. 4,771,131; and European PatentApplication No. 88/402,115.5. Subsequently, other Coleopteran-activestrains have been isolated as described in PCT patent publications WO91/00791 and WO 90/09445. The Bt tenebrionis strain which carries theColeopteran-active cryIIIa gene has been reported to kill a variety ofColeoptera. However, according to Slaney et al (1992) the toxin encodedby this gene was found to be much less effective to Diabrotica larvaethan to the Colorado potato beetle, Leptinotarsa decemlineata.Diabrotica was found to have a poor ability to bind the CryIIIA toxin.

Objects and Summary of the Invention

An object of the present invention is to provide two new Bt strains ofpathotype C; i.e., the BTS02584B and BTS02584C strains and variants ofthese strains.

Yet another object of the present invention is to provide sporulatedcultures of BTS02584B and BTS02584C which possess insecticidal activityand can therefore be formulated into insecticidal compositions againstColeoptera in general and especially against Diabrotica species,Agelastica alni, Hypera postica, Hypera brunneipennis, Halticatombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum,Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata (Colorado potato beetle) and morepreferably against D. undecimpunctata undecimpunctata (Western spottedcucumber beetle), D. undecimpunctata howardi (Southern corn rootworm),D. barberi (Northern corn rootworm) and D. virgifera virgifera (Westerncorn rootworm), which are major pests of economically important cropssuch as corn.

Yet another object of the present invention is to provide aninsecticidal composition against Coleoptera and a method for controllingColeoptera with the insecticidal composition by contacting insects withthe insecticidal composition that comprises BTS02584B and/or BTS02584Cstrains, BTS02584B and/or BTS02584C sporulated cultures and/or theactive insecticidal component(s).

Detailed Description of the Preferred Embodiments of the Invention

As used herein the term variant means a strain which differs from otherrelated strains in a specified or unspecified way but retains the sameinsecticidal activity as the related strain. Variants can be made byconventional means including mutation by ultraviolet light sources, byuse of chemicals such as nitrosoguanidine and the like.

More specifically the present invention relates to two new strains ofBacillus thuringiensis with significant insecticidal activity againstinsects of the family Chrysomelidae, particularly Diabrotica andLeptinotarsa spp. such as the Colorado potato beetle, Lepinotarsadecemlineata and the Western corn rootworm, Diabrotica virgiferavirgifera. Strains BTS02584B and BTS02584C are isolated from grain dustfound in the Phillipines using the usual methods known in the art(Travers et al., 1987). These strains typically grow in elongated chainsof bacterial cells that give the liquid culture medium a flocculentappearance. In these long strands, spores are readily recognized underthe microscope as opaque oval bodies, while the crystals are recognizedas dense bipyramidal bodies. Upon higher magnification, the crystalsseem to be composed of two pyramidal bodies linked together in thebipyramidal crystal. The strains can be grown in any medium such as T-3agar and the like, but it is preferable to grow the strains in C2medium.

Insect bioassays show a marked insecticidal activity of both strains andvariants thereof to Leptinotarsa decemlineata and Diabrotica virgiferavirgifera, but show no insecticidal activity to Lepidoptera.

The insecticidal active component found in these strains of the presentinvention is heat labile, is not insecticidal towards Lepidoptera andpreliminary evidence indicates that the component is larger than 10 kD.It is believed that the insecticidal active component is a protein or apolypeptide-like compound which is present in sporulated cultures of theBt strain.

An insecticidal, particularly anti-Coleopteran, composition of thepresent invention can be formulated in a conventional manner usingsporulated cultures of the BTS02584B and/or BTS02584C strains or theirinsecticidal component(s) as active ingredients with suitable carriers,diluents, emulsifiers, dispersants and/or attractants. The insecticidalcomposition can be formulated as a wettable powder, pellets, granules,dust or as a liquid formulation with aqueous or non-aqueous solvents asa foam, gel, suspension, concentrate and the like. The concentration ofthe BTS02584B and/or BTS02584C strains and/or their insecticidalcomponents in a composition depends upon the nature of the formulationand its intended mode of use. Generally, an insecticide composition ofthe present invention can be used to protect a potato or corn field forabout 2 to 4 weeks against Coleoptera after the composition is applied.For more extended protection (e.g., for an entire growing season),additional amounts of the composition should be applied periodically.

A method for controlling insects, particulalrly Coleoptera, inaccordance with the present invention preferably comprises applying(e.g., spraying) to a locus (area) to be protected an insecticidallyeffective amount of the BTS02584B and/or BTS02584C strains or theirinsecticidally active components. The locus to be protected can include,for example, the habitat of the insect pests or growing vegetation or anarea where vegetation is to be grown.

To obtain the insecticidal component(s) of the BTS02584B and BTS02584Cstrains, cells of the BTS02584B and BTS02584C strains can be grown in aconventional manner on a suitable culture medium and then lysed usingconventional means such as enzymatic degradation, detergents or thelike. The insecticidal component(s) can then be separated and purifiedby standard techniques such as chromatography, extraction,electrophoresis or the like. If the insecticidal component(s) is provento be a polypeptide it can be purified and sequenced by conventionalmethods as described in European Patent Publication Nos. 193,259 and458,819. After the sequence is isolated it can be placed into anappropriate vector system and recombinantly produced according to themethods described in Molecular Cloning, A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory Press, (1989).

The BTS02584B and BTS02584C cells also can be harvested and then appliedintact, either dead or alive, preferably dried, to the locus to beprotected. In this regard, it is preferred that purified BTS02584Band/or BTS02584C strains (either dead or alive) be utilized. It is mostpreferable to use a cell mass that contains 90.0 to 99.9% purifiedBTS02584B and BTS02584C.

The BTS02584B and/or BTS02584C sporulated cultures or their insecticidalcomponent(s) can be formulated in an insecticidal composition in avariety of ways using any number of conventional additives, wet or dry,depending upon the particular use. Additives may include, for example,wetting agents, detergents, stabilizers, adhering agents, spreadingagents, extenders and the like. Examples of such compositions includepastes, dusting powders, wettable powders, granules, baits, aerosolcompositions and the like. Other Bt cells, toxins and insecticidallyeffective toxin portions and other insecticides, fungicides, biocides,herbicides, fertilizers and the like can be utilized in the compositioncontaining the BTS02584B and/or BTS02584C sporulated cultures or theirinsecticidal component(s) to provide advantages or benefits. Such aninsecticidal composition can be prepared in a conventional manner andthe amount of BTS02584B and/or BTS02584C cells and/or insecticidalcomponent(s) employed depends upon a variety of factors such as theinsect pest targeted, the composition used, the type of area to whichthe composition is to be applied and the prevailing weather conditions.Furthermore, BTS02584B or BTS02584C sporulated cultures may be usedseparately or can be mixed together to form the active ingredient of theinsecticidal composition. Generally, the concentration of BTS02584B orBTS02584C insecticidal component(s) is at least about 0.1% by weight ofthe formulation to about 100% by weight of the formulation andpreferably from about 0.15% to about 0.8% of the formulation.Specifically for corn rootworm, application is preferably made to theroots.

In practice, some insects can be fed the BTS02584B and/or BTS02584Csporulated cultures, their insecticidally effective component(s) ormixtures thereof in the protected area; that is in the area wheresporulated cultures or insecticidally effective component(s) have beenapplied. Alternatively, some insects can be fed intact live cells of theBTS02584B and/or BTS02584C strains or variants thereof, so that theinsects ingest the insecticidal component(s) of these strains and sufferdeath or severe damage.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given, it being understoodthat the same are intended only as illustrative and in nowiselimitative.

EXAMPLE 1 Characterization of the BTS02584B and BTS02584C strains

The BTS02584B and BTS02584C strains were deposited under the provisionsof the Budapest Treaty on Jun. 14, 1993 at the Belgian CoordinatedCollections of Microorganisms--Collection Laboratorium voorMicrobiologie Belgium ("BCCM-LMG"), University of Gent, K.Ledeganckstraat 35, B-9000 Gent, Belgium. BTS02584B was deposited underaccession number LMG P-14025, BTS02584C under accession number LMGP-14026.

The BTS02584B and BTS02584C strains were cultivated at 28° C. on C2medium (containing: 10 g/l glucose, 5 g/l casamino acid, 2 g/l peptone,2 g/l yeast extract, 0.247 g/l MgCl₂.6H₂ O; 0.058 g/l MnCl₂.4H₂ O; 0.1g/l CaCl₂ ; 0.005 g/l ZnSO₄.7H₂ O; 0.005 g/l CuSO₄.7H₂ O; 1.619 g/l NH₄Cl; 0.0005 g/l FeSO₄.7H₂ O; after autoclaving the following was added:3.11 g/l KH₂ PO₄ and 4.66 g/l K₂ HPO₄). For long term storage, an equalvolume of a spore crystal suspension with an equal volume of 50%glycerol was mixed and stored at -70° C. or lyophilized as a sporesuspension. For sporulation, the use of liquid C2 medium was utilizedfor 72 hours at 28° C., followed by storage at 4° C. During theirvegetative phase, the BTS02584B and BTS02584C strains can also growunder facultative anaerobic conditions, but sporulation only occursunder aerobic conditions.

After cultivating on C2 Agar ("CA", C2 medium containing 2% Agar, DifcoLaboratories, Detroit, Mich., U.S.A.) for one day, colonies of theBTS02584B and BTS02584C strains formed opaque white colonies withirregular edges. Cells of the strains sporulated after three dayscultivation at 28° C. on CA. The crystal proteins produced duringsporulation were packed in crystals in the BTS02584B and BTS02584Cstrains. Furthermore, these strains typically grew in elongated chainsof bacterial cells that gave the liquid culture medium a flocculentappearance. In these long strands, spores were readily recognized underthe microscope as opaque oval bodies, while the crystals were recognizedas dense bipyramidal bodies. Upon higher magnification, the crystalsseemed to be composed of two pyramidal bodies linked together in thebipyramidal crystal.

Moreover, possibly because of this auto-agglutination, the Bt serotypecould not be determined on the BTS02584B and BTS02584C strains byconventional serotyping methods as conducted by the WHO CollaboratingCenter for Entomopathogenic Bacillus (M. Lecadet).

EXAMPLE 2 Characteristics of the BTS02584B and BTS02584C insecticidalcomponent(s)

The BTS02584B and BTS02584C strains were grown for 72 hours at 28° C. onC2 medium. After sporulation, the sporulated cultures were harvested bycentrifugation to pellet the cells and the spores and were thensuspended in a tap water-Triton X-100 solution (75 μl TX-100 per 200 mltap water; TX-100 from Sigma, St. Louis, U.S.A.), that is non-toxic toDiabrotica. The suspensions of these sporulated cultures were testedagainst coleopteran and lepidopteran insects and were found to be highlyand specifically toxic for the tested Coleoptera. Furthermore, theinsecticidal component(s) in these suspensions was tested for heatinactivation for 10 min. at 95° C. The results of the heat inactivationtest are set forth in Table 1. The specificity of the activity and thethermolability are strong indications of the proteinaceous nature of theBTS02584B and BTS02584C insecticidal component(s).

Dialysis of the suspension of sporulated cultures of Bt strain BTS02584Bindicated that the insecticidal component had a molecular weight of over10 kD. The insecticidal activity was retained after prolonged dialysisusing membranes having a molecular weight cut-off point of about 10 kD.This is another indication of the proteinaceous nature of the component.

EXAMPLE 3 Insecticidal activity of the BTS02584B and BTS02584C strains

The BTS02584B and BTS02584C strains were grown for 72 hours at 28° C. onC2 medium. After sporulation, the sporulated cultures were harvested ina tap water-Triton X-100 solution (75 μl TX-100 per 200 ml tap water;TX-100 from Sigma, St. Louis, U.S.A.) that is non-toxic to Diabrotica.The suspensions of these sporulated cultures were tested againstDiabrotica virgifera virgifera, Leptinotarsa decemlineata and someselected Lepidoptera.

For Diabrotica virgifera v. assays, pieces of corn leaves of one weekold plants (size: 1 cm²) were dipped in a suspension of sporulatedBTS02584B or BTS02584C cultures and were dried in a laminar flowcupboard. When dry, the leaflets were placed on agar (2%) in 24multiwell plates. On each leaflet 4 neonate Diabrotica virgiferavirgifera larvae were placed; 24 larvae were used per bioassay. After 2days the leaflets were removed and fresh corn leaves were placed in thewells. After four days the dead and living larvae were counted. Theresults on D. virgifera virgifera are shown in Table 1.

For Leptinotarsa decemlineata assays, potato leaves were dipped eitherin a suspension of sporulated BTS02584B or BTS02584C cultures and thenair dried for two hours. Colorado potato beetle larvae of the firstinstar were placed on the treated leaves, and mortality of the larvaewas determined after three days. These results were compared with themortality of larvae fed leaves treated with spore-crystal mixtures ofBTS1 (B. thuringiensis BTS1 from DSM accession no 4288) which was usedas a reference strain. The results obtained with the Colorado potatobeetle larvae are shown in Table 2.

The assays showed that the suspension of the BTS02584B and BTS02584Csporulated cultures caused Diabrotica and Leptinotarsa larvae to stopfeeding after about one day and die within a few days. In addition,Table 1 shows that boiling the suspension for 10 minutes resulted in asignificant decrease in insecticidal activity towards D. virgifera. Thisindicates that the insecticidally effective compound is heat-labile.

Furthermore, suspensions of sporulated cultures of both strainsBTS02584B and BTS02584C were shown to have no toxicity towards thetested Lepidoptera of Plutella xylostella, Heliothis virescens andSpodoptera littoralis which were not killed and developed normally.These Lepidoptera were tested in diet application assays. Artificialdiet was dispensed in wells of Costar 24-well plates. 50 μl of a sampledilution (in phosphate buffered saline--Bovine serum albumin buffercontaining per liter of distilled water: 8.7 g NaCl, 22.5 g Na₂ HPO₄.2H₂O, 2 g KH₂ PO₄, 0.1% BSA) was applied on the surface of the diet anddried in a laminar air flow. Two larvae were placed in each well usedper sample dilution (neonate larvae were used, except for Plutella inwhich third instar larvae were used). These data show the specificity ofthe BTS02584B and BTS02584C insecticidal component(s) towardsColeoptera, and more specifically to Diabrotica and Leptinotarsa.

EXAMPLE 4 Further Insecticidal activity of the BTS02584B and S02584Cstrains

Following the procedure set forth in Example 3, BTS02584B and BTS02584Cstrains are used to test the insecticidal activity on the followinginsect pests:

Diabrotica undecimpunctata undecimpunctata, Diabrotica undecimpunctatahowardi, Diabrotica barberi, Agelastica alni, Hypera brunneipennis,Hypera postica, Haltica tombacina, Anthonomus grandis, Tenebrio molitor,Triboleum castaneum, Dicladispa armigera, Trichispa serica, Oulemaoryzae, Colaspis brunnea, Lissorhorptrus oryzophilus, Phyllotretacruciferae, Phyllotreta striolata, Psylliodes punctulata, Entomoscelisamericana, Meligethes aeneus, Ceutorynchus species, Psylliodeschrysocephala and Phyllotreta undulata.

Similar results are obtained as indicated in Tables 1 and 2.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

                  TABLE 1                                                         ______________________________________                                        Diabrotica virgifera virgifera Larvae                                                                Concentration                                                     Experiment  Spores-                                                Strain     Number      crystal/ml  Mortality                                  ______________________________________                                        BTS0258B   1           >10.sup.9   71%                                        Control    1           0           12%                                        (Triton-                                                                      water)                                                                        BTS0258B   2           4.4 × 10.sup.9                                                                      96%                                                               4.4 × 10.sup.9                                                                      88%                                                               10.sup.9    88%                                                               10.sup.9    92%                                        Control    2           0           0                                          BTS02584B  3           3.4 × 10.sup.9                                                                      96%                                                               3.4 × 10.sup.9                                                                      92%                                                               10.sup.9    79%                                                               10.sup.9    92%                                        BTS02584C  3           3.4 × 10.sup.9                                                                      88%                                                               10.sup.9    79%                                        Control    3           0            4%                                        BTS02854B  4           10.sup.9    79%                                                               10.sup.9    83%                                                               10.sup.9    83%                                        BTS02584C  4           10.sup.9    100%                                                              10.sup.9    88%                                                               10.sup.9    79%                                                               10.sup.9    100%                                       Control    4           0            4%                                        BTS02584B  5           10.sup.9    88%                                                               10.sup.9    92%                                                               10.sup.9    92%                                        BTS02584B  5           10.sup.9    33%                                        (boiled)               10.sup.9    21%                                                               10.sup.9    54%                                        Control    5           0            8%                                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Leptinotarsa decemlineata larvae                                                                     Concentration                                                     Experiment  Spores-                                                Strain     Number      crystal/ml  Mortality                                  ______________________________________                                        BTS02584B  1           10.sup.9     87%                                       BTS1.sup.a 1           10.sup.9    100%                                       Control    1           0            6%                                        (buffer)                                                                      BTS02584C  2           10.sup.9    100%                                                              10.sup.9    100%                                       BTS1.sup.a 2           10.sup.9    100%                                                              10.sup.9    100%                                       Control    2           0           0                                          ______________________________________                                         .sup.a Leptinotarsaactive BTS1 strain of DSM accession number 4288       

References

Ellar et al, in "Fundamental and Applied aspects of InvertebratePathology", ed. Samson, R. A., Vlak, J. M. and Peters, D., pp. 7-10,Wageningen, Foundation of the fourth International Colloquim ofInvertebrate Pathology, 1986.

Levine and Oloumi-Sadeghi, Annu. Rev. Entomol. 36, 229-55, 1991.

Metcalf, R. L., Foreword in "Methods for the Study of Pest Diabrotica",pp. vii-xv, eds. Krysan, J. L. and Miller, T. A., Springer-Verlag, NewYork, 1986.

Slaney, A. C. et al, Insect Biochem. Molec. Biol. 22, 9-18, 1992.

Travers et al., Applied & Environmental Microbiology., vol. 53,1263-1266, 1987.

What is claimed is:
 1. A biologically pure culture of Bacillusthuringiensis LMG P-14025.
 2. A biologically pure culture of Bacillusthuringiensis LMG P-14026.